Spirochetes are a unique and diverse group of helical-shaped bacteria that include the pathogens Treponema pallidum (syphilis) and Borrelia burgdorferi (Lyme disease). The incidence of both Lyme disease and syphilis has increased markedly over the past several years resulting in considerable morbidity. The pathogenesis of spirochetes is reflected, in part, by their unique mode of motility. For example, non-motile spirochetes are less able to penetrate endothelial cell monolayers, suggesting an active role of motility in penetration. Because this unique motility has been implicated as a factor in the pathogenesis of diseases caused by spirochetes, this proposal focuses on the molecular genetic analysis of motility proteins of T. pallidum and Treponema phagedenis. The major organelles responsible for spirochete motility are the periplasmic flagella, which consist of the filament and the hook-basal body (HBB) apparatus. In contrast to the filament genes, nothing is known about the structural genes comprising the HBB of spirochetes. The first aim of this proposal is to clone and sequence the gene encoding the hook protein of T. pallidum using a probe developed from T. phagedenis. The DNA and predicted amino acid sequence will be compared to the T. phagedenis hook gene (flgE) which may reveal putative pathogen-specific sequences in addition to highly conserved regions that are critical for motility of spirochetes. Although this work is designed to ultimately understand T. pallidum motility, the nonpathogenic T. phagedenis is an essential model for studying the motility genes of treponemes because it is cultivable and readily manipulated in vitro. Several lines of evidence suggest that T. phagedenis flgE is part of a large operon that encodes additional motility proteins including structural components of the HBB. The major goal of this proposal is to elucidate the sequence, organization, and function of the motility genes and regulatory elements located on this operon in T. phagedenis and the homologous operon in the pathogenic T. pallidum. This work will initially involve cloning and sequencing of the DNA upstream from flgE to identify putative motility genes and regulatory elements. The organization and transcription of the T. phagedenis operon will be characterized using Northern blotting and RNase protection assays. The newly identified T. phagedenis motility genes will be cloned into an Escherichia coli protein expression plasmid and purified recombinant protein will be isolated. Antisera will be generated to these motility proteins for determination of the location of each protein using immunoelectron microscopy and Western blotting. To initiate gene regulation studies, motility mutants will be generated using chemical mutagenesis and analyzed using the above techniques together with dark field and electron microscopy. After the identification and extensive molecular characterization of the genes involved in treponemal motility, the broad long term objectives are to contribute to the knowledge of spirochete motility and to determine if motility proteins are useful for vaccine or diagnostic purposes.